This invention relates to an efficient genetic transformation method for zoysiagrass (Zoysia japonica Steud.), also called Korean grass. The present invention can be applicable to other closely related turfgrass species. The present invention also provides the transgenic zoysiagrass plant that is resistant to herbicide and the experimental methods and processes for the generation of such a herbicide-resistant zoysiagrass.
The present invention is to provide an efficient Agrobacterium tumefaciens-mediated genetic transformation method for zoysiagrass (Zoysia japonica Steud.), also known as Korean grass, the optimised media composition and culture condition that greatly influence the transformation efficiency, and the transgenic zoysiagrass plant that has herbicide resistance and associated experimental procedures for the development of such a transgenic zoysiagrass with the bialaphos resistance gene (bar).
Zoysiagrass is one of the most important species of turfgrass and widely cultivated in the Far-Eastern Asia, including Korea, Japan, and Eastern area of China, as well as in the temperate zone worldwide. The cultivation area of zoysiagrass is rapidly expanding in USA and other countries in recent years, primarily due to its extraordinary characteristics such as the resistance to drought and the capacity to rapidly recover from traffic damage. In addition, it grows well in poor soil in virtually all climates. Due to these useful traits, it is widely used for golf courses, athletic fields, roadsides, home gardens, and riverbanks. As its market size is rapidly growing, customers demand new varieties with improved resistance to pathogens, herbicides, and various environmental stresses. Until recently, classical breeding methods have been mainly employed to develop such new traits in turfgrass. However more and more laboratories and institutes are striving to apply molecular biological methods to genetically engineer turfgrass (Inokuma et al. 1998; Park and Ahn 1998) since it is now possible to develop or modify useful traits in a predictable way by using these methods.
Although most monocots are not readily infected by Agrobacterium tumefaciens, efficient A. tumefaciens-mediated transformation systems have been successfully recently developed for a few species of the Gramineae family, such as rice (Hiei et al. 1994; Rashid et al. 1996), maize (Ishida et al. 1996), and wheat (Cheng et al. 1997). Unfortunately, however, such A. tumefaciens-mediated transformation methods have not been established yet for turfgrass (Chai and Sticklen 1998) except for the bentgrass (Yu et al. 2000). Especially, the genetic transformation of zoysiagrass is further hampered by some additional technical problems. For example turfgrass seed germination rate is very low, and production of regenerable callus is difficult (Asano 1989).
Callus morphology is closely related to plant regenerability as has been proven in various plant species (Armstrong and Green 1985; Ke and Lee 1996; Luo and Jia 1998). Recently, we established an efficient callus induction and plant regeneration system for zoysiagrass (Bae et al. 2001), which was filed as U.S. Pat. Ser. No. 09/915,294 on Jul. 27, 2001.
According to the present invention, it is now possible to genetically transform zoysiagrass with a gene of interest by A. tumefaciens-mediated infection.
Some potential target traits for the genetic transformation of zoysiagrass include improved ground-covering capacity, tolerance to traffic injury, rapid recovering after damage, resistance to biotic and abiotic stress, engineered (accelerated or delayed) growth rate, and shade avoidance. Of particular interests is to engineer growth rate and/or shade avoidance so that the maintenance cost for watering and mowing can be drastically reduced.
With recent rapid accumulation of molecular biological technology and establishment of efficient tissue culture and genetic transformation systems in plants, any gene of agronomical importance can now be readily introduced into any desired plants with aims to enhance crop yield and quality and environmental adaptability. In the present invention, we provide an efficient Agrobacterium tumefaciens-mediated zoysiagrass transformation method and a transgenic zoysiagrass plant with herbicide resistance.
As used herein, the term xe2x80x9cgenetic transformationxe2x80x9d refers to a procedure to introduce a gene(s) or genetic material(s) into a higher plant of interest in a predictable way. The gene or genetic material is stably integrated into the plant genome and transmitted through generations.
The present invention relates to a reliable Agrobacterium tumefaciens-mediated transformation method for zoysiagrass that is to be routinely used for the genetic transformation of zoysiagrass or closely related turfgrass species.
The present invention also provides a method for genetically transforming the zoysiagrass (Zoysia genus) comprising the steps of: i) inducing and growing the calli of zoysiagrass on modified MS medium containing various hormones; ii) infecting the calli of zoysiagrass with Agrobacterium cells to introduce bialaphos resistance bar gene; iii) co-cultivating calli of zoysiagrass and Agrobacterium cells in co-cultivation medium containing acetosyringone without 2,4-dichlorophenoxyacetic acid and calcium; iv) eliminating Agrobacterium cells from co-cultivation medium; and v) regenerating the transgenic zoysiagrass.
Further, Agrobacterium cells possess the pGPT-HB transforming vector, and type 3 calli (which is defined in our previous application Ser. No. 09/915,294) are co-cultivated in co-cultivation medium for 5xcx9c7 days.
Also, the present invention provides a transgenic zoysiagrass by above method, which is stably transformed with the bar gene under control of plant-specific promoter. The particular promoter is ubiquitin promoter from maize.
Critical media components or culture conditions evaluated in the invention include callus type, co-cultivation period, 2,4-D (2,4-dichlorophenoxyacetic acid), CaCl2, and acetosyringone. The highest transformation efficiency was obtained when type 3 callus was co-cultivated on a 2,4-D-free medium for 5xcx9c7 days. In addition, removal of calcium and inclusion of 30xcx9c70 mg/liter acetosyringone during co-cultivation greatly enhanced the transformation efficiency.
When the optimized transformation protocol was used for zoysiagrass transformation with the bar gene, up to 20.5% of plated shoots on the selection medium exhibited herbicide-resistant.
Also, provided in the invention is the transgenic zoysiagrass plant with herbicide resistance. The transgenic zoysiagrass could survive even after 5 gram/liter herbiace solution was sprayed every day for 2 weeks and eventually grew to maturity, whereas control plants stopped growing and died when treated under the same experimental condition.
Therefore, the present invention further provides the transgenic zoysiagrass callus with herbicide resistance (deposited under accession No. KCTC-10076BP).
Said callus was deposited at Korea Collection for Type Cultures, #52 Oun-Dong, Yusong-ku, Taejon, Seoul, 305-333, Republic of Korea with accession number KCTC-10076BP on Sep. 21, 2001 under Budapest treaty.